Prostate cancer is a major form of cancer in U.S. males. Prostate specific antigen (PSA) serum levels provide a good marker for prostate cancer, but assay of total serum PSA alone cannot distinguish early cancer from a number of other prostatic diseases. Numerous invasive and expensive procedures may be required for diagnosis. Preliminary results of the applicant indicate that PSA exists in different forms in semen and in serum and that different PSA forms are found in the sera of different patients. This proposal requests funding to establish a new independent research area for the applicant, in which the various forms of PSA will be identified, assayed in a large number of patients with borderline elevations of total PSA, and correlated to the diagnosis. The long-term goal is to provide the biochemical and immunochemical basis for clinical assays of forms of PSA that distinguish early prostate cancer from other conditions that cause modestly elevated PSA. It is hypothesized: a) that PSA as an enzyme may have a different role in prostate cancer than in benign conditions of the prostate or prostatitis; b) that the enzyme PSA is secreted in inactive zymogen form, activated under particular pathophysiological conditions, and inactivated by proteolytic cleavage or by combining with available protease inhibitors to form complexes; c) that the local environment and the type of epithelial cells that secrete PSA differ in prostate cancer compared to other syndromes; d) that the activators, proteolytic enzymes and inhibitors to which PSA is exposed differ in cancer compared to other syndromes; e) that the time elapsed between secretion of PSA and its translocation to the blood differs in cancer versus other syndromes; f) that the spectrum of enzymes and inhibitors in prostatic fluid is different from that of blood; and g) that all of the above lead to the generation of different forms of PSA in prostate cancer from those found in other syndromes. The applicant's previous experience in elucidating the molecular forms and inhibitors of other enzymes can be readily applied to the identification and measurement of different forms of PSA in patients. The specific aims include: 1) isolation of PSA from semen, characterization of its reactivity with various protease inhibitors, and preparation of standard PSA-inhibitor complexes; 2) development of sandwich ELISA assays for PSA complexes with any significant inhibitors, including alpha1-antichymotrypsin, protein C inhibitor, alpha2-macroglobulin, and others that may be identified, using standard complexes; 3) identification of different forms of PSA in semen and in blood by immunoblot analysis and sequencing studies; 4) development of antibodies and ELISA-assays specific for particular forms or sequences of PSA; 5) application of immunoblot analysis, sandwich ELISAS and other assays which are developed to large numbers of patient sera, especially those in the borderline PSA range; and 6) correlation of the spectrum and levels of different PSA forms to patient diagnosis to establish clinical usefulness of the assays. The proposed studies also provide novel basic knowledge about PSA and its inhibitors that may help to understand the physiological function and regulation of this enzyme.